R E S E A R C H Open AccessInvestigation of Chlamydiaceae in semen and cauda epididymidis and seroprevalence of Chlamydophila abortus in breeding bulls Ann-Charlotte Karlsson1,2*, Stefan
Trang 1R E S E A R C H Open Access
Investigation of Chlamydiaceae in semen and
cauda epididymidis and seroprevalence of
Chlamydophila abortus in breeding bulls
Ann-Charlotte Karlsson1,2*, Stefan Alenius1, Camilla Björkman1, Ylva Persson3, Stina Englund4
Abstract
Background: Reproductive disorders associated with chlamydial infection have been reported worldwide in cattle and there are indications of potential venereal transmission
Methods: Semen samples from 21 dairy bulls and cauda epididymidis tissue samples from 43 beef bulls were analysed for chlamydial agent by real-time polymerase chain reaction (PCR) including an internal amplification control (mimic) Additionally, presence of antibodies against Chlamydophila (Cp.) abortus among the bulls was investigated with the commercial Pourquier® ELISA Cp abortus serum verification kit
Results: No chlamydial agent was detected by PCR in either the semen samples or in the tissue samples
Additionally, no antibodies against Cp abortus were detected
Conclusions: The results suggest that Cp abortus is very rare, or absent in Swedish bulls and thus the risk for venereal transmission of chlamydial infection through their semen is low However, because Chlamydophila spp infection rates seem to differ throughout the world, it is essential to clarify the relative importance of transmission
of the infection through semen on cattle fertility
Background
Bovine chlamydiosis has been associated with several
disease manifestations [1] Reproductive disorders such
as sporadic abortions and reduced fertility, linked with
chlamydial infection have been reported from Germany
[2,3], Great Britain [4], Italy [5], Japan [6], Switzerland
[7], Taiwan [8] and the USA [9] In Sweden, the
inci-dence of abortion in cows is low However, reproductive
disorders and infertility are major causes of culling but
are often difficult to be diagnosed Chlamydial infection
in bulls may be the cause to some of these problems
[10] Experimental studies have shown that the bacteria
can be excreted in semen of inoculated bulls and rams
[11] and isolation of the agent from semen of naturally
infected bulls and rams has been reported [12-14] The
vaginal mucosa in sheep and uterine mucosa in cattle
are susceptible to infection [15,16] and transmission of
chlamydial agent by experimentally infected semen to heifers and sheep has been demonstrated [17,18] The two species Chlamydophila (Cp.) abortus and Cp pecorum are known to infect cattle and are suggested to
be ubiquitous [9,19] Moreover, Cp psittaci infections in cattle have been reported [20,21] All three species have been identified in bull semen [22,23] Cp abortus is the cause of Ovine Enzootic Abortion (OEA), the major infectious cause of abortion and lamb loss with great economic losses in many sheep-producing countries [24] Cp pecorum has foremost been associated with polyarthritis, encephalitis and inapparent intestinal infection, and the impact by Cp psittaci in ruminants is yet to be investigated
Each year about 80 top-ranked performance-tested yearling beef bulls are sold all over Sweden, mainly to pedigree breeders, after six months of testing at the only performance testing station in the country These per-formance-tested bulls represent the best-documented beef bulls with the highest impact on the breeding pro-gramme in Sweden and are therefore important poten-tial transmitters of Chlamydophila spp by venereal
* Correspondence: ann-charlotte.karlsson@vetinst.no
1 Division of Ruminant Medicine and Veterinary Epidemiology, Department of
Clinical Sciences, Faculty of Veterinary Medicine and Animal Science,
Swedish University of Agricultural Sciences (SLU), Box 7054, SE-750 07,
Uppsala, Sweden
© 2010 Karlsson et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
Trang 2route Additionally, artificial insemination (AI) is
per-formed yearly on more than 95% of the approximately
400,000 Swedish dairy cows [25] As there is a
possibi-lity of transmission of Chlamydophila spp via this
route, it is important to determine whether breeding
bulls are infected through screening of semen before AI
in order to minimize this risk The aim of this study
was to investigate the presence of chlamydial agent in
semen and in tissue of cauda epididymidis and to
esti-mate the seroprevalence of Cp abortus in Swedish bulls
Methods
Animals and samples
Beef bulls
This study comprises samples from a subset of 166 beef
bulls from 124 herds from different parts of Sweden
that were taken to the only performance testing station
in Sweden in September 2002 On arrival the bulls were
approximately six months old They were divided into
groups, based on breed and body weight and placed in
ten adjacent semi-outdoor pens under the same roof
The bulls were weighed every second week throughout
the testing period (September-March) and at the end of
the period, an individual growth index was calculated
Bulls with fast growth rates were sold at livestock
auc-tion and bulls with growth indexes below the threshold,
stated by the breeders’ organisations, were either
slaugh-tered or returned to their owners In total, 43 of the
beef bulls that were sent to slaughter were included in
this study (23 Charolaise, 7 Hereford, 6 Simmental, 3
Aberdeen Angus, 3 Limousine and 1 Blonde
d’Aqui-taine) The daily growth rates of these bulls were
some-what lower than the bulls sold at auction, but were still
higher than the growth rates of non-tested beef sires in
Sweden [26] Because of co-operation with another
study and their definite criteria [27], only bulls with
clinically normal reproductive organs and scrotal
cir-cumference above 30 cm were included
Testes and epididymides were removed at the time of
slaughter and immediately put in a container with
crushed ice and transported refrigerated to the laboratory
where they arrived the next day An incision (approx 1.5
cm long and 0.5 cm deep) was made with a scalpel blade
in the middle, distal part of the cauda epididymides [28],
and a sample of 0.5 × 0.5 cm was taken and stored at
-70°C until used for DNA preparation In addition, blood
samples for serum preparation were taken on arrival (6
month of age) and at departure (1 year of age) from the
testing station Sera were stored at -20°C
Dairy bulls
Semen samples (0.2 ml payettes) and sera from 21 dairy
bulls about 1 year old (Swedish Holstein and Swedish
Red) in service, were submitted to the laboratory from
one of the only two semen producing companies in
Sweden, Svensk Avel http://www.vikinggenetics.com Semen samples were stored at -70°C prior to prepara-tion for analysis by real-time polymerase chain reacprepara-tion (PCR) Sera for serology were stored at -20°C until analysed
Detection of Chlamydiaceae by real-time polymerase chain reaction
DNA was extracted from semen and cauda epididymidis samples for PCR analysis using a High Pure Template Preparation kit, following manufacturer’s instructions (Roche Diagnostics, Basel, Switzerland) and stored at -20°C Analyses were performed using a Chlamydiaceae-specific real-time PCR protocol developed by Everett and others [29], targeting the 23S ribosomal DNA Briefly, the primers used were TQF (5’-GAA AAG AAC CCT TGT TAA GGG AG-3’) and TQR (5’-CTT AAC TCC CTG GCT CAT CAT G-3’) The sequence of the fluorescent FAM-labelled probe was 5’-CAA AAG GCA CGC CGT CAA C-3’
An internal amplification control (mimic) was con-structed and used to detect false negative PCR results,
as previously described [30] The primers used in the
AAC CCT TGT TAA GGG AGC CAT GTA CCC
CTG GCT CAT CAT GGA TCC ACA CGG AGT ACT TGC-3’) The sequence of the ROX-labelled mimic probe used in real-time PCR was 5’-CCG ACA GGA TGC AGA AGG AGA TCA-3’
The 25-μl PCR mixture comprised 2.5 μl of 10× PCR-buffer II (Applied Biosystems, Foster City, CA, USA), 2.5 mM MgCl2, 0.2 mM of each of the four dNTP, 0.15
μM of each of the primer TQF and TQR, 0.25 μl (1.25 U) of AmpliTaq Gold DNA polymerase (Applied
were placed in a Rotor-Gene 3000 (Corbett Research, Cambridge, UK) and amplification was performed according to the protocol of Everett and others [29] The results were analysed with the Rotor-Gene software version 5.0
The sensitivity of the PCR was estimated to one inclu-sion forming unit (IFU) per PCR by spiking semen and tissue samples prior to DNA extraction with ten-fold dilutions of Cp abortus (inactivated strain S26/3 in ori-ginal concentration of 3 × 108 IFU/ml, kindly provided
by D Longbottom, Moredun Research Institute, UK) Detection of antibodies to Cp abortus
For detection of antibodies the Pourquier® ELISA Chla-mydophila abortus serum verification kit (Montpellier, France) was applied The ELISA uses a recombinant fragment of an 80-90 kDa polymorphic outer membrane protein and detects antibodies against Cp abortus The assay was used according to the manufacturer’s instruc-tion with S/P% values≥ than 100 as positive for cattle
Trang 3All 21 semen and 43 cauda epididymidis samples were
negative in the PCR The internal amplification control
(mimic) worked well for all samples analysed
None of the 21 and 43 paired-sera from dairy and beef
bulls, respectively, were positive in the antibody
detec-tion assay Most samples were clustered well below the
cut-off value 100 Only six samples had S/P% above 20,
where 42 was the highest value
Discussion
In this study we found no presence of chlamydial agent
in any semen or cauda epididymidis tissue samples, i.e
all samples were negative by real-time PCR This is in
concordance with an Austrian study [31] where neither
Cp abortus nor Cp pecorum was detected in 273 semen
samples from bulls at five AI centres On the other
hand, the results contradict those reported from other
investigations performed in apparently healthy bulls In
Lithuania as much as 29.8% of 47 tested bulls had
chla-mydial agent in their semen, as judged by PCR [13], and
chlamydiae were detected by immunofluorescence in
14.3% of 42 bovine ejaculates from the Czech republic
[32] In German and Swiss investigations of semen
sam-ples, 9.2% and 6.6%, respectively, were found positive by
PCR [22,23]
The sensitivity of the PCR assay was estimated to 1
IFU per PCR with no indication of potential inhibitory
factors In a previous investigation of cows from dairy
herds with reproductive disorders we identified positive
specimens, including vaginal swabs, placenta and milk
when using the same PCR assay [33] Moreover, several
positive specimens from different organs in pigs and
placentae in sheep (unpublished data) as well as
con-junctival and nasal swabs from cats [34] have been
demonstrated by the same PCR at our laboratory Those
samples were handled and stored in a similar way as in
the present study Therefore, the test is considered
robust and to have a high sensitivity and specificity
All sera were negative in the Cp abortus ELISA assay
with values far below the cut-off value The specificity
of the test has been reported to be 100% when used to
analyse Scottish sheep documented free of Cp abortus
[35] and 90% when sera from New Zealand, a country
free from Cp abortus, were analysed [36] The
sensitiv-ity were estimated to 91% and 80%, respectively, when
analysing sera from experimentally Cp abortus infected
sheep [35,36]., and it can, hence, not be excluded that
some of our sera were positives but not detected by the
test However, the fact that all the beef bulls, which
came from as many as 124 different herds from all over
Sweden, were still seronegative after they had been
housed together for six months, in adjacent pens under
the same roof, indicates that Cp abortus is not present
in Swedish beef cattle herds Moreover, the absence of seropositives among the analysed dairy bulls indicates that Cp abortus is very rare, or absent, in Swedish bulls These results are in agreement with a previous study in Swedish dairy cows where only 2 out of 525 sera were positive in the same ELISA and only Cp pecorum were confirmed in vaginal swabs [33]
Conclusions
This study suggest the risk for venereal transmission of chlamydial infection through Swedish bull semen is low However, because Chlamydophila spp infection rates seem to differ throughout the world, it is essential to clarify the relative importance of transmission of the infection through semen on cattle fertility
Acknowledgements The authors wish to thank Maj Hjort at the National Veterinary Institute (SVA), for performing the serological analyses and for assistance with the DNA preparations This study was supported by the Swedish Farmer ’s Foundation for Agricultural Research and the Programme for Infection Biology at the Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences It was part of the EU research collaboration COST 855.
Author details
1
Division of Ruminant Medicine and Veterinary Epidemiology, Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences (SLU), Box 7054, SE-750 07, Uppsala, Sweden 2 Department of Animal Health, Section for Farm Animal Health and Welfare, National Veterinary Institute, Pb 750 Sentrum, N-0106 Oslo, Norway.3Department of Animal Health and Antimicrobial Strategies, Section of Farm animals, National Veterinary Institute (SVA), SE-751 89, Uppsala, Sweden.4Department of Animal Health and Antimicrobial Strategies, Section of Antibiotics, National Veterinary Institute (SVA), SE-751
89, Uppsala, Sweden.
Authors ’ contributions ACK participated in the design and coordination of the study, drafted and rewrote the manuscript, carried out the PCR and interpreted the results SE implemented the PCR systems, constructed the mimic and interpreted the results CB and SA participated in the design and coordination of the study.
YP sampled and wrote about the beef bulls All authors have been involved
in revising the manuscript.
Competing interests The authors declare that they have no competing interests.
Received: 14 December 2009 Accepted: 13 January 2010 Published: 13 January 2010 References
1 Storz J: Overview of animal diseases induced by chlamydial infections Microbiology of Chlamydia Barron, AL Florida: CRC Press, Inc 1988, 167-192.
2 Wehrend A, Failing K, Hauser B, Jager C, Bostedt H: Production, reproductive, and metabolic factors associated with chlamydial seropositivity and reproductive tract antigens in dairy herds with fertility disorders Theriogenology 2005, 63:923-930.
3 Wittenbrink MM, Schoon HA, Bisping W, Binder A: Infection of the bovine female genital tract with Chlamydia psittaci as a possible cause of infertility Reprod Dom Anim 1993, 28:129-136.
4 Holliman A, Daniel RG, Parr JG, Griffiths PC, Bevan BJ, Martin TC, Hewinson RG, Dawson M, Munro R: Chlamydiosis and abortion in a dairy
Trang 45 Cavirani S, Cabassi CS, Donofrio G, De Iaco B, Taddei S, Flammini CF:
Association between Chlamydia psittaci seropositivity and abortion in
Italian dairy cows Prev Vet Med 2001, 50:145-151.
6 Nabeya M, Kaneko K, Ogino H, Nakabayashi D, Watanabe T, Murayama J,
Hayashi K, Fukushi H, Yamaguchi T, Hirai K, Inaba Y, Matumoto M: Abortion
in Japanese cows caused by Chlamydia psittaci Vet Microbiol 1991,
29:261-265.
7 Pospischil A, Thoma R, von Bomhard W, Reitt K, Cantieni J,
Zimmermann DR, Polkinghorne A: [Abortion in cattle caused by
Chlamydia psittaci] Schweiz Arch Tierheilkd 2002, 144:467-472.
8 Wang FI, Shieh H, Liao YK: Prevalence of Chlamydophila abortus infection
in domesticated ruminants in Taiwan J Vet Med Sci 2001, 63:1215-1220.
9 Kaltenboeck B, Hehnen H-R, Vaglenov A: Bovine Chlamydophila spp.
infection: do we underestimate the impact on fertility? Vet Res Comm
2005, 29:1-15.
10 Storz J, Carroll EJ, Ball L, Faulkner LC: Isolation of a psittacosis agent
(Chlamydia) from semen and epididymis of bulls with seminal vesiculitis
syndrome Am J Vet Res 1968, 29:549-555.
11 Storz J, Carroll EJ, Stephenson EH, Ball L, Eugster AK: Urogenital infection
and seminal excretion after inoculation of bulls and rams with
chlamydiae Am J Vet Res 1976, 37:517-520.
12 Amin AS: Comparison of polymerase chain reaction and cell culture for
the detection of Chlamydophila species in the semen of bulls,
buffalo-bulls, and rams Vet J 2003, 166:86-92.
13 Domeika M, Ganusauskas A, Bassiri M, Fröman G, Mårdh P-A: Comparison
of polymerase chain reaction, direct immunofluorescence, cell culture
and enzyme immunoassay for the detection of Chlamydia psittaci in
bull semen Vet Microbiol 1994, 42:273-280.
14 Gautam R, Purohit VD: Isolation of Chlamydia psittaci from genitalia of
healthy exotic and crossbred service rams Indian Journal of Animal
Sciences 2001, 71:731-734.
15 Papp JR, Shewen PE: Pregnancy failure following vaginal infection of
sheep with Chlamydia psittaci prior to breeding Infect Immun 1996,
64:1116-1125.
16 Wittenbrink MM, Schoon HA, Schoon D, Mansfeld R, Bisping W:
Endometritis in cattle experimentally induced by Chlamydia psittaci.
Zentralbl Veterinarmed B 1993, 40:437-450.
17 Appleyard WT, Aitken ID, Anderson IE: Attempted venereal transmission of
Chlamydia psittaci in sheep Vet Rec 1985, 116:535-538.
18 Bowen RA, Spears P, Storz J, Deidel GE Jr: Mechanisms of infertility in
genital tract infections due to Chlamydia psittaci transmitted through
contaminated semen J Infect Dis 1978, 138:95-98.
19 DeGraves FJ, Gao D, Hehnen HR, Schlapp T, Kaltenboeck B: Quantitative
Detection of Chlamydia psittaci and C pecorum by High-Sensitivity
Real-Time PCR Reveals High Prevalence of Vaginal Infection in Cattle J
Clin Microbiol 2003, 41:1726-1729.
20 Borel N, Thoma R, Spaeni P, Weilenmann R, Teankum K, Brugnera E,
Zimmermann DR, Vaughan L, Pospischil A: Chlamydia-related abortions in
cattle from Graubunden, Switzerland Vet Pathol 2006, 43:702-708.
21 Cox HU, Hoyt PG, Poston RP, Snider TG, Lemarchand TX, O ’Reilly KL:
Isolation of an avian serovar of Chlamydia psittaci from a case of bovine
abortion J Vet Diagn Invest 1998, 10:280-282.
22 Kauffold J, Henning K, Bachmann R, Hotzel H, Melzer F: The prevalence of
chlamydiae of bulls from six bull studs in Germany Anim Reprod Sci
2007, 102:111-121.
23 Teankum K, Pospischil A, Janett F, Brugnera E, Hoelzle LE, Hoelzle K,
Weilenmann R, Zimmermann DR, Gerber A, Polkinghorne A, Borel N:
Prevalence of chlamydiae in semen and genital tracts of bulls, rams and
bucks Theriogenology 2007, 67:303-310.
24 Aitken ID: Chlamydial abortion Diseases of Sheep Edinburgh:
BlackwellMartin WB, Aitken ID , 3 2000, 81-86.
25 SJV: Number of cattle in December 2004 Swedish official statistics 2004, JO
23 SM 0501.
26 Eriksson S: Genetic aspects of calving, growth, and carcass traits in beef
cattle PhD thesis Swedish University of Agricultural Sciences, Department
of Animal Breeding and Genetics 2003.
27 Persson Y: Breeding Soundness Evaluation of Young Beef Bulls PhD thesis
Swedish University of Agricultural Sciences, Department of Clinical Sciences
2007.
28 Gustafsson B, Crabo B: Some morphological characteristics of bull spermatozoa at different levels of the epididymis Nord Vet Med 1971, 23:114-118.
29 Everett KDE, Hornung LJ, Andersen AA: Rapid detection of the Chlamydiaceae and other families in the order Chlamydiales: three PCR tests J Clin Microbiol 1999, 37:575-580.
30 Englund S, Bolske G, Ballagi-Pordany A, Johansson KE: Detection of Mycobacterium avium subsp paratuberculosis in tissue samples by single, fluorescent and nested PCR based on the IS900 gene Vet Microbiol 2001, 81:257-271.
31 Petit T, Spergser J, Aurich J, Rosengarten R: Examination of semen from bulls at five Austrian artificial insemination centres for chlamydiae and mollicutes Vet Rec 2008, 162:792-3.
32 Veznik Z, Pospisil L, Svecova D, Kummer V, Canderle J, Diblikova I, Maskova J: Chlamydiae in reproductive disorders and pathology of reproductive organs in man and animals Reprod Dom Anim 1996, 31:595-600.
33 Godin AC, Björkman C, Englund S, Johansson KE, Niskanen R, Alenius S: Investigation of Chlamydophila spp in dairy cows with reproductive disorders Acta Vet Scand 2008, 50:39.
34 Bölske G, Ström Holst B, Stillesjö A, Englund S: Detection of Chlamydiaceae
in Swedish cats with a real-time PCR with internal control Diagnosis, pathogenesis and control of animal Chlamydioses The fourth annual workshop of COST Action 855, Animal Chlamydioses and Zoonotic Implications: 3-5 September 2006; Edinburgh Meigle Colour Printers LtdLongbottom D, Rocchi M 2006, 74-75.
35 Vretou E, Radouani F, Psarrou E, Kritikos I, Xylouri E, Mangana O: Evaluation
of two commercial assays for the detection of Chlamydophila abortus antibodies Vet Microbiol 2007, 123:153-161.
36 McCauley LM, Lancaster MJ, Young P, Butler KL, Ainsworth CG: Comparison
of ELISA and CFT assays for Chlamydophila abortus antibodies in ovine sera Aust Vet J 2007, 85:325-328.
doi:10.1186/1751-0147-52-2 Cite this article as: Karlsson et al.: Investigation of Chlamydiaceae in semen and cauda epididymidis and seroprevalence of Chlamydophila abortus in breeding bulls Acta Veterinaria Scandinavica 2010 52:2.
Publish with Bio Med Central and every scientist can read your work free of charge
"BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright
Submit your manuscript here: BioMedcentral